TOPOLOGICALLY CONSTRAINED DOMAINS OF SUPERCOILED DNA IN EUKARYOTIC CELLS

The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comp arison with a panel of irradiated supercoiled plasmid DNAs, Loss of su percoiling in plasmids was determined by gel electrophoresis and in ce lls by nucleoid flow cytometry, Comparison of dose-response data for p lasmid relaxation with that obtained from SQ-20B cells enabled a direc t estimation of supercoil target size in these cells, Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kb p), and R6K (38 kbp) were used and analyzed under the same exposure co nditions as nucleoid DNA, Two sizes of topologically closed domains we re found in nucleoids of 0.51 +/- 0.17 Mbp and 1.34 +/- 0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with functio n, cells mere exposed to the DNA topoisomerase II inhibitor, VP16 and the G(1)/S cell cycle blocking agent mimosine. A 1h exposure to VP16 w as effective in reducing DNA synthesis which was associated with a par allel increase in nucleoid supercoiling. Addition of the G(1) > S inhi bitor mimosine enhanced both responses, It is concluded that chromosom es and interphase nuclei are organized into at least two sizes of topo logically constrained domains of DNA which may have functional relevan ce to the control and execution of DNA synthesis.